Enpp2 haploinsufficiency induces an eye-open-at-birth phenotype in the DBA/2 background

Autotaxin, encoded by the Enpp2 gene, produces lysophosphatidic acid (LPA), which exerts numerous biological functions via its cognate receptors. Enpp2 null mutant mice die by embryonic day 9.5 owing to aberrant vascular development in the yolk sac, preventing analysis after that period. In this study, we found that Enpp2 heterozygous mice in the DBA/2 genetic background showed an eye-open-at-birth phenotype at high frequency, caused by failure of eyelid closure during the embryonic stage. Notably, wildtype pups from the Enpp2 heterozygous dam showed the phenotype, although at lower frequency, suggesting that maternal LPA affects the embryonic development.

(A) Eye appearance of Enpp2 +/+ or Enpp2 +/-mice at P28.About 80% of the Enpp2 +/-mice showed smaller eyes and corneal opacity after 3 consecutive backcrosses to DBA/2J.(B) Table showing the numbers and percentages of mice with corneal opacity at P28, after 4 consecutive backcrosses to DBA/2J.The numbers and percentages of the offsprings with EOB are classified and shown according to the parental genotypes.(C) Eye-open phenotype of Enpp2 +/-embryos and neonates.The eyelids were open in both the Enpp2 +/+ and the Enpp2 +/-embryos at E14.5.In the Enpp2 +/+ embryos, the eyelids were closed at E16.5 and remained closed at P7, whereas in the Enpp2 +/-embryos, the eyelids remained open throughout the embryonic and neonatal periods.(D) Hematoxylin-eosin-stained transverse slices of the eyes of Enpp2 +/+ or Enpp2 +/-embryos at E17.5.Eyelid epithelial extension was impaired in the Enpp2 +/-embryos (black arrows).Scale bar, 300 µm.(E) In situ hybridization of Ennp2.At E10.5, no signal was observed.At E12.5, when the eyelids were extended, strong Enpp2 expression was observed in the mesenchymal cells just beneath the extending epithelium, which persisted until E15.5.Scale bar, 50 µm.(F) LacZ staining of Enpp2 +/+ or Enpp2 +/-embryos.At E11.5, the signals were detected in the eyelids and whisker hair follicles of the Enpp2 +/-embryos.The signals in the eyelid region remained strong until E15.5.The signals were not observed in the Enpp2 +/+ embryo.Scale bar, 400 µm.
In this study, we examined the impact of genetic background on the Enpp2 mutation because in general, differences in genetic background can weaken or strengthen the phenotypes of KO mice (Montagutelli, 2000).We backcrossed Enpp2 heterozygous mice, maintained on a mixed genetic background of C57BL/6N and 129SvJ, to various inbred strains, including C57BL/6N, 129SvJ, BALB/cA, C3H/HeJ, and DBA/2J.After backcrosses to DBA/2J for 3 consecutive generations, we noticed for the first time that some postnatal mice developed corneal opacity (Fig. 1A): none of the mice backcrossed to other inbred strains developed such a phenotype nor showed other visible changes.The frequency of corneal opacity at postnatal day 28 (P28) was about 80% in the Enpp2 +/-mice in the offspring backcrossed to DBA/2J for 4 consecutive generations (Fig. 1B).Intriguingly, a small number (8.9%) of the WT mice also showed a similar phenotype (Fig. 1B).To eliminate the possibility that a spontaneous mutation affecting eye development occurred during backcrossing, we conducted an independent backcross to DBA/2J and found that the same phenotype appeared at a similar frequency.When examined separately by each parent's genotype, the WT pups with corneal opacity were observed in those born from Enpp2 +/-females mated with WT males, but not in those born from WT females mated with Enpp2 +/-males (Fig. 1B).These data imply that the maternal genotype influences the phenotypic appearance of the offspring.
Previous studies have demonstrated a correlation between corneal opacity and an eye-open-at-birth (EOB) phenotype.In mice, eyelid development begins with the groove extending over the cornea around E12, and the eyelid primordia meet and fuse around E16.5 (Findlater et al., 1993).The eyelids remain fused until P12-14 when they reopen as a result of apoptosis of the eyelid tissues.When the eyelids are not closed at the embryonic stage, corneal injury induces inflammation and opacity after birh (Nunomura et al., 2021;Wu et al., 2016).To determine whether the ocular abnormalities observed in Enpp2 +/-mice are caused by EOB, we examined the eye development of embryos generated by mating WT female mice with Enpp2 +/-male mice to minimize maternal effects.The eyelid primordia were formed in all the embryos at E12.5 to E14.5 (Fig. 1C).At E16.5, the eyelids had already fused in the WT embryos, but they remained open in the Enpp2 +/-embryos (Fig. 1C).At P7, the eyelids remained closed in the WT mice, whereas the Enpp2 +/-mice showed opened eyelids and corneal opacity (Fig. 1C).Histological analysis of transverse slices of the eye at E17.5 revealed that the epithelial sheets over the eye were not formed in the Enpp2 +/-embryos, whereas no apparent abnormalities were observed in the lens, retina, or cornea (Fig. 1D).These data indicate that ocular abnormalities observed in postnatal Enpp2 +/-mice resulted from a failure of eyelid extension and closure during the embryonic stage.The corneal opacity seems to be a secondary effect resulting from the injury caused by the absence of eyelids and loss of physical protection.
Finally, we examined the Enpp2 expression in the eye region of the embryos.Initially, we detected Enpp2 mRNA using in situ hybridizaton.At E10.5, Enpp2 signals were undetectable around the eye (Fig. 1E).At E12.5, strong signals were detected in the mesenchymal cells, especially beneath the extending epithelium, but not in the epithelial cells of the eyelids.The expression remained detectable until E15.5.Next, we investigated Enpp2 expression by means of wholemount LacZ staining, as the LacZ gene was knocked in the Enpp2 KO allele.The signal was weakly detected around the eye at E11.5 and had become stronger at E15.5 (Fig. 1F).The signals were not detected in the WT mice, suggesting the specificity of the lacZ reaction (Fig. 1F).
Our data indicate that Enpp2 is expressed in the mesenchymal cells of eyelids when the eyelids extend and that decreased Enpp2 expression induces the EOB phenotype in the DBA/2J background, suggesting that Enpp2 is required for the proliferation and/or migration of mesenchymal cells in the eyelids.Why the EOB phenotype appears only in the DBA/2J background is unknown.However, given that DBA/2J is a model for congenital experimental glaucoma (Turner et al., 2017), the strain may have unidentified susceptibility to eye diseases associated with maldevelopment of the anterior eye segment.Interestingly, heterozygosity of the dam partially induces the EOB phenotype in their WT offspring, suggesting that LPA in the dam somehow affects the eye development of their embryos.Although it also remains unknown whether LPA is supplied from the matermal circulation to the embryo, it is possible that an unidentified LPA transporter in the placenta facilitates transport of LPA as amino acids and fatty acids are transported (Brett et al., 2014) and extracellular LPA can activate peroxisome proliferator-activated receptor (PPARγ) in monocytic cells intracellularly across cell membranes (McIntyre et al., 2003).Investigating how maternal LPA impacts embryonic development will be necessary in future.

Animals
All experimental procedures involving animals were approved by the Animal Care and Use Committee of the University of Tsukuba and performed in accordance with its guidelines.Enpp2 KO mice (B6;129SvJ-Enpp2 tm1Mmas ) were generated by use of homologous recombination in 129/Ola-derived ES cells (Koike et al., 2009).The mutant mice were backcrossed to C57BL/6N, 129SvJ, BALB/cA, C3H/HeJ, and DBA/2J, which were purchased from CLEA Japan.Offspring were genotyped by use of PCR using the primers 5'-CTGCTGAAACTTAATGCACTGGAC-3' (Enpp2 forward), 5'-TGTGTAAGTCAGGGAACAACTCTG-3' (Enpp2 reverse), and 5'-TGCTCCAGACTGCCTTGGGAAAAG-3' (neo).Noon of the day when a vaginal plug was observed was taken as embryonic day 0.5 (E0.5).Embryos were taken after mice were sacrificed by means of cervical dislocation.

Histology
For histological examination, mouse embryos were fixed with 4% PFA in PBS.Subsequently, the fixed embryos were processed and embedded in paraffin by use of a tissue processor ASP200 (Leica).Slices of 4-µm thickness were then cut with a microtome RM2145 (Leica) and stained with hematoxylin-eosin (Muto Pure Chemicals).